Proteins (e.g., peptides, oligopeptides, and polypeptides) are responsible for most of the activities of a cell, such as catalysis, communication, defense, movement, and transport. The underlying basis of a protein's biological activity is its amino acid sequence and/or its conformation. Accordingly, the biologically active portion of a protein should remain essentially intact and in its biologically functional conformation. The advancements in genetic engineering techniques for protein expression have led to the development of methods for the controlled expression of both native and foreign proteins in various systems in a form that maintains the biological activity of the proteins. Such genetically engineered, controlled expression systems can result in higher protein yield due to the expression of properly-folded, stable proteins where inactivation and degradation of the proteins is reduced as a result of the ability to control protein expression.
Although a number of different types of expression systems have been developed, in various organelles of host cells such as microorganisms, eukaryotic cells, including fungi, yeast, and mammalian cells, insect cells, etc., an expression system for the controlled expression of proteins utilizing stability factors of nuclear origin to regulate the expression of proteins in plastids has not previously been developed. Plastids are organelles responsible for photosynthesis and are commonly classified as chloroplasts, leucoplasts, amyloplasts, or chromoplasts. Plastids can differentiate or redifferentiate between these forms.
In one embodiment, a method for preparing an expression system for inducing the production of a protein in the plastid of a cell is provided. The method comprises the steps of introducing a first nucleic acid into the nucleus of a cell wherein the first nucleic acid encodes an inducible promoter, operatively linking the first nucleic acid to a second nucleic acid to form a recombinant nucleic acid wherein the second nucleic acid encodes a stability factor, wherein the introduction of an inducer or the removal of a repressor induces the expression of the stability factor, wherein the expressed stability factor associates in the plastid with an untranslated region of an mRNA stabilized by the stability factor and transcribed from a third nucleic acid, wherein the third nucleic acid is either native to the plastid or is foreign to the plastid and wherein the third nucleic acid encodes the protein, and wherein expression of the mRNA results in the production of the protein.
In another illustrative embodiment, a method for preparing an expression system for repressing the expression of a plastid protein in the plastid of a cell is provided. The method comprises the steps of introducing a first nucleic acid into the nucleus of a cell wherein the first nucleic acid encodes a repressible promoter, operatively linking the first nucleic acid to a second nucleic acid to form a recombinant nucleic acid wherein the second nucleic acid encodes a stability factor, wherein the introduction of a repressor or the removal of an inducer represses the expression of the stability factor, and wherein the repression of the expression of the stability factor results in the repression of expression of an mRNA stabilized by the stability factor and transcribed from a third nucleic acid, wherein the third nucleic acid is either native to the plastid or is foreign to the plastid and wherein the third nucleic acid encodes the protein, and wherein the expression of the protein is repressed.
In still another illustrative aspect, a method for expressing a plastid protein in the plastid of a cell is provided. The method comprises the steps of contacting the cell with an inducer or treating the cell under conditions that result in the removal of a repressor, wherein the inducer or the repressor associates with a first nucleic acid in the nucleus, wherein the first nucleic acid encodes an inducible promoter, wherein the first nucleic acid is operatively linked to a second nucleic acid to form a recombinant nucleic acid and wherein the second nucleic acid encodes a stability factor, expressing the stability factor, introducing the stability factor into the plastid wherein the stability factor associates in the plastid with an untranslated region of an mRNA to stabilize the mRNA wherein the mRNA is transcribed from a third nucleic acid which is either native to the plastid or is foreign to the plastid and wherein the third nucleic acid encodes the protein, expressing the mRNA, and producing the protein in the plastid.
In another embodiment, a method for repressing the expression of a plastid protein in the plastid of a cell is provided. The method comprises the steps of contacting the cell with a repressor or treating the cell under conditions that result in the removal of an inducer, wherein the repressor or the inducer associates with a first nucleic acid in the nucleus of the cell, wherein the first nucleic acid encodes a repressible promoter, wherein the first nucleic acid is operatively linked to a second nucleic acid to form a recombinant nucleic acid, and wherein the second nucleic acid encodes a stability factor, repressing the expression of the stability factor wherein the stability factor associates in the plastid with an untranslated region of an mRNA to stabilize the mRNA wherein the mRNA is transcribed from a third nucleic acid which is either native to the plastid or is foreign to the plastid and wherein the third nucleic acid encodes the protein, repressing the expression of an mRNA, and repressing the expression of the protein.
In still another embodiment, a system for expressing a plastid protein in the plastid of a recombinant host cell is provided. The system comprises an exogenously added inducer that induces the expression of a nuclear protein, the recombinant host cell wherein the nucleus of the recombinant host cell comprises a recombinant nucleic acid, wherein the recombinant nucleic acid comprises a first nucleic acid operatively linked to a second nucleic acid to form the recombinant nucleic acid, wherein the first nucleic acid encodes an inducible promoter and wherein the second nucleic acid encodes a stability factor, and the plastid comprising a third nucleic acid that is either native to the plastid or foreign to the plastid wherein the third nucleic acid encodes the expressed plastid protein wherein the expression of the mRNA encoding the plastid protein is controlled by the stability factor.
In another illustrative embodiment, a system for repressing the expression of a plastid protein in the plastid of a recombinant host cell is provided. The system comprises an exogenously added repressor that represses the expression of a nuclear protein, the recombinant host cell wherein the nucleus of the recombinant host cell comprises a recombinant nucleic acid, wherein the recombinant nucleic acid comprises a first nucleic acid operatively linked to a second nucleic acid to form the recombinant nucleic acid, wherein the first nucleic acid encodes a repressible promoter and wherein the second nucleic acid encodes a stability factor, and the plastid comprising a third nucleic acid that is either native to the plastid or foreign to the plastid wherein the third nucleic acid encodes the expressed plastid protein and wherein the expression of the mRNA encoding the protein is controlled by the stability factor.
In another embodiment, a method for stimulating the production of hydrogen gas by expressing a plastid protein in the plastid of a cell is provided. The method comprises the steps of contacting the cell with an inducer or treating the cell under conditions that result in the removal of a repressor, wherein the inducer or the repressor associates with a first nucleic acid in the nucleus, wherein the first nucleic acid encodes an inducible promoter, wherein the first nucleic acid is operatively linked to a second nucleic acid to form a recombinant nucleic acid and wherein the second nucleic acid encodes a stability factor, expressing the stability factor, introducing the stability factor into the plastid wherein the stability factor associates in the plastid with an untranslated region of an mRNA to stabilize the mRNA wherein the mRNA is transcribed from a third nucleic acid which is either native to the plastid or is foreign to the plastid wherein the third nucleic acid encodes the protein, expressing the mRNA, producing the protein in the plastid, and producing hydrogen gas.
In yet another embodiment, a method for inhibiting the production of hydrogen gas by repressing the expression of a plastid protein in the plastid of a cell is provided. The method comprises the steps of contacting the cell with a repressor or treating the cell under conditions that result in the removal of an inducer, wherein the repressor or the inducer associates with a first nucleic acid in the nucleus of the cell, wherein the first nucleic acid encodes a repressible promoter, wherein the first nucleic acid is operatively linked to a second nucleic acid to form a recombinant nucleic acid, wherein the second nucleic acid encodes a stability factor, repressing the expression of the stability factor wherein the stability factor associates in the plastid with an untranslated region of an mRNA to stabilize the mRNA wherein the mRNA is transcribed from a third nucleic acid which is either native to the plastid or is foreign to the plastid and wherein the third nucleic acid encodes the protein, repressing the expression of the mRNA, repressing the expression of the protein, and inhibiting the production of hydrogen gas.
In still another embodiment, a method for stimulating the production of hydrogen gas by inducing and repressing the expression of a plastid protein in the plastid of a cell is provided. The method comprises the steps of sequentially i) contacting the cell with an inducer or treating the cell under conditions that result in the removal of a repressor and ii) contacting the cell with the repressor or treating the cell under conditions that result in the removal of the inducer, wherein the inducer or the repressor associates with a first nucleic acid in the nucleus, wherein the first nucleic acid encodes an inducible promoter, wherein the first nucleic acid is operatively linked to a second nucleic acid to form a recombinant nucleic acid and wherein the second nucleic acid encodes a stability factor, sequentially expressing and repressing the expression of the stability factor, wherein the stability factor associates in the plastid with an untranslated region of an mRNA to stabilize the mRNA wherein the mRNA is transcribed from a third nucleic acid which is either native to the plastid or is foreign to the plastid wherein the third nucleic acid encodes the protein, sequentially expressing and repressing the expression of the mRNA, producing the protein in the plastid, and producing hydrogen gas.
In any of the above-described embodiments, the first nucleic acid can be operatively linked to the second nucleic acid to form the recombinant nucleic acid prior to introducing the recombinant nucleic acid into the nucleus, the cell can have an inoperative copy or can be missing a copy or a homolog of the second or the third nucleic acid, the cell can be a plant cell or an algal cell, the plastid can be selected from the group consisting of a chloroplast, a leucoplast, a amyloplast, an etioplast, an elaioplast, and a chromoplast, the inducible promoter can have at least 90% sequence similarity to the Cyc6 promoter, and the third nucleic acid can encode a gene that has at least 90% sequence similarity to the psbD gene.
In any of the above-described embodiments, the inducer or repressor can be a chemical or an environmental condition where the chemical can be copper and where the environmental condition can be reduction in the concentration of oxygen to a predetermined level, the inducer can be applied and removed for a plurality of cycles wherein a cycle comprises applying and removing the inducer, the protein can be a protein involved in photosynthesis or in the production of hydrogen gas, the protein can be selected from the group consisting of a pharmaceutical agent, an industrial enzyme, an enzyme involved in chloroplast maturation or degradation, and a nutraceutical where the pharmaceutical agent is selected from the group consisting of an antibody, a vaccine antigen, and an antimicrobial agent, or other defense products for the host cell and the stability factor can be selected from the group consisting of Nac2 and Mbb1. In another illustrative embodiment, the second nucleic acid can code for a translational activating factor, such as, for example, Tbc2 or Tca1.
In another embodiment of the invention, a system and method for regulating the expression or repression of native or foreign genes in plastids is provided. In one embodiment, the invention relates to an expression system employing a nuclear-encoded chloroplast transcription factor, Nac2, the expression of which is regulated by an inducible promoter of the Cyc6 gene. In another embodiment, induction of Nac2 expression by an inducer (i.e., an agent or alteration of an environmental condition), such as low levels of oxygen, causes the expression of the psbD gene in the chloroplast. In yet another embodiment, an agent or environmental condition, such as removal of copper, that causes induction of the Cyc6 promoter, also causes expression of the psbD gene. In another illustrative embodiment, repression of the Nac2 gene by a repressor (i.e., an agent or alteration of an environmental condition), such as high levels of oxygen, results in no or reduced expression of the psbD gene. In a related embodiment, an agent or alteration in an environmental condition that represses the inducible Cyc6 promoter also causes reduced expression of the psbD gene.
In other illustrative embodiments, the invention relates to the inducible expression of or the repression of a foreign gene in the chloroplast whereby replacement of the psbD gene in the chloroplast with a foreign gene facilitates inducible expression of or the repression of the foreign gene in the chloroplast by regulation of Nac2 expression.
In another embodiment, the invention relates to a method of producing hydrogen gas in the chloroplast through regulation of the psbD gene by Nac2 expression or repression of expression of Nac2. In this illustrative aspect of the invention, environmental conditions that facilitate the induction and repression of the Nac2 gene (e.g., reducing the level of oxygen to induce expression and elevating the level of oxygen to repress expression), resulting in the oscillating induction and repression of psbD gene expression, result in a reduction in the rate of photosynthesis and a resulting reduction in the concentration of oxygen. In this embodiment, the reduction in the concentration of oxygen facilitates the production of hydrogen. Thus, in one embodiment, the invention relates to a method of producing hydrogen gas by regulating the oscillating induction and repression of Nac2 and psbD gene expression.
In yet another embodiment, the invention relates to a method for enhancing the hydrogen-generating system through the recombinant expression of other genes in the chloroplast, for example, hydrogenases and repression of other recombinant or native proteins, such as phosphoribulose kinase.
In still another embodiment an apparatus for the production of hydrogen is provided. The apparatus comprises a first vessel configured to hold a cell culture in a substantially oxygen-depleted environment, a first pump in fluid communication with the first vessel and configured to pump a medium into the first vessel at a predetermined rate, and a measuring device coupled to the first vessel and configured to measure an amount of hydrogen produced by the cell culture.
In this embodiment, the first pump can be configured to pump the amount of medium into the first vessel at a rate substantially equal to the rate of growth of the cell culture, the first pump can comprise a peristaltic pump, the cell culture can comprise a cy6Nac2.49 culture, the measuring device can comprise a mass spectrometer, the apparatus can further comprise an agitation device coupled to the first vessel and operable to agitate the cell culture, the agitation device can comprise a magnetic stir bar, the apparatus can further comprise a second vessel configured to hold an amount of the medium, wherein the first pump is fluidly coupled to the second vessel and configured to pump the medium from the second vessel at the predetermined rate, the apparatus can further comprise a third vessel in fluid communication with the first vessel and configured to hold an overflow of the medium from the first vessel, and the apparatus can further comprise a filter and a second pump in fluid communication with the third vessel and the second vessel, the second pump being configured to pump an amount of the medium from the third vessel, through the filter, and into the second vessel.